Cyclophilins are intracellular receptor proteins for cyclosporin A (CsA), a widely recognized immunosuppressive agent. Cyclophilin proteins exhibit peptidyl-prolyl cis-trans isomerase (PPIase) activity, which catalyzes cis-trans isomerization of peptide bonds preceding proline, and play functional roles in chaperoning and protein folding. The immunosuppressive activity for which CsA is so well known does not directly result from inhibiting cyclophilin activity. Rather, a CsA-cyclophilin A complex inhibits the Ca2+/calmodulin-dependent phosphatase calcineurin, thereby suppressing T-cell proliferation by interfering with downstream signal transduction. (See J. Lee and S. S. Kim, Journal of Experimental & Clinical Cancer Research, 2010, 29:97; J. W. Elrod and J. D. Molkentin, Circulation Journal, 2013, 77:1111; C. Piot, et al., New England Journal of Medicine, 2008, 359:473.)
CsA binds to most members of the cyclophilin family, including cyclophilin A (CypA) and cyclophilin D (CypD). CypA is found in the cytosol, while CypD is a mitochondrial matrix protein. The role ascribed to CypD in the mitochondrial matrix is to modulate the mitochondrial permeability transition pore (MPTP), a non-specific high-conductance channel in the inner mitochondrial membrane. MPTP opening increases inner mitochondrial cell membrane permeability, allowing an influx of cytosolic molecules into the mitochondrial matrix. This influx increases the matrix volume and disrupts the outer mitochondrial membrane, which may lead to cell death. MPTP regulation by CypD appears to provide a physiologic Ca2+ release mechanism for proper control of mitochondrial metabolism. For example, in the case of myocardial infarction, MPTP opening may be triggered by calcium overload and excessive reactive oxygen species (ROS) at the time of reperfusion, resulting in metabolic alterations (e.g., collapse of mitochondrial membrane potential, uncoupling of the respiratory chain, efflux of pro-apoptotic factors, and hydrolysis of ATP), which may lead to cardiomyocyte death. (See J. W. Elrod, et al., supra; C. Piot, et al., New England Journal of Medicine, 2008, 359:473; S. Y. Kim, et al., Cell Death and Disease, 2014, e1105).
CsA has been reported to limit ischemia-reperfusion injury under experimental conditions. In a small proof-of-concept trial on the effects of CsA on reperfusion injury in acute myocardial infarction, CsA reduced infarct size when administered at the time of reperfusion. These findings led to the hypothesis that CsA-induced inhibition of MPTP opening occurs by preventing the calcium-induced interaction of CypD with an MPTP component. (See C. Piot, et al., supra).
CsA has also been reported to interact with CypD to inhibit MPTP opening and ameliorate neuronal cell death against ischemic injury in the central nervous system, and has been studied in the context of ischemic retinal injury, specifically, ischemic injury in response to elevated intraocular pressure (TOP) (See S. Kim, et al., Cell Death and Disease, 2014, e1105.) IOP presents risks for retinal ganglion cell (RGC) death and optic nerve degeneration in retinal ischemia and glaucoma. Investigations into the mechanism for ischemic retinal injury induced by acute increases in IOP have revealed that acute IOP elevation significantly upregulates CypD protein expression in ischemic retina, which may, in turn, facilitate MPTP opening, increase mitochondrial vulnerability and trigger cell death in the ischemic retina. CsA was found to prevent CypD upregulation and to promote RGC survival against ischemic injury by preventing mitochondrial alteration. CsA-mediated CypD inhibition may thus have promising therapeutic potential for protecting RGCs against ischemic injury mediated mitochondrial dysfunction. (Id.)
CsA (CAS Registry Number: 59865-13-3) is a naturally occurring fungal metabolite and the first identified member of the cyclosporin family of poly-N-methylated cyclic undecapeptides having the following structure:

CsA consists of 11 amino acids and can be further represented as follows:

wherein:
MeBmt is (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine;
αAbu is L-α-aminobutyric acid;
Sar is sarcosine;
MeLeu is N-methyl-L-leucine;
Val is L-valine;
Ala is L-alanine;
DAla is D-alanine; and
MeVal is N-methyl-L-valine.
The numbers 1-11 are used to designate each of the eleven amino acids. Thus, MeBMT is the amino acid at position 1, while sarcosine is the amino acid at position 3. In certain instances, the description herein may refer to the amino acid side chain at any one of positions 1-11. The carbon to which the amino acid side chain is attached is referred to as the alpha (α) carbon.
Cyclosporin B is identical to CsA, except that αAbu is replaced by L-alanine.
Cyclosporin C is identical is identical to CsA, except that αAbu is replaced by L-threonine.
Cyclosporin D is identical to CsA, except that αAbu is replaced by L-valine.
CsA, which binds to most members of the cyclophilin family, is not specific for mitochondrial CypD. (J. W. Elrod et al., supra; C. Piot et al., supra).
There remains a need for CypD-selective inhibitors.
The present invention relates to the surprising discovery of non-immunosuppressive analogs of CsA that are potent inhibitors of CypD.